Measurement of Oxidatively Induced DNA Damage in Caenorhabditis elegans with High-Salt DNA Extraction and Isotope-Dilution Mass Spectrometry.

Caenorhabditis elegans is used extensively as a medical and toxicological model organism. However, little is known about background levels of oxidatively induced DNA damage in the nematode or how culturing methods affect DNA damage levels. The tough C. elegans cuticle makes it challenging to extract genomic DNA without harsh procedures that can artifactually increase DNA damage. Therefore, a mild extraction protocol based on enzymatic digestion of the C. elegans cuticle with high-salt phase-separation of DNA has been developed and optimized. This method allows for efficient extraction of >50 µg DNA using a minimum of 250000 nematodes grown in liquid culture. The extracted DNA exhibited acceptable RNA levels (<10% contamination), functionality in polymerase chain reaction assays, and reproducible DNA fragmentation. Gas chromatography/tandem mass spectrometry (GC-MS/MS) with isotope-dilution measured lower lesion levels in high-salt extracts than in phenol extracts. Phenolic extraction produced a statistically significant increase in 8-hydroxyguanine, a known artifact, and additional artifactual increases in 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 4,6-diamino-5-formamidopyrimidine, and 8-hydroxyadenine. The high-salt DNA extraction procedure utilizes green solvents and reagents and minimizes artifactual DNA damage, making it more suitable for molecular and toxicological studies in C. elegans. This is, to our knowledge, the first use of GC-MS/MS to measure multiple 8,5'-cyclopurine-2'-deoxynucleosides in a toxicologically important terrestrial organism.

Aflatoxin-Guanine DNA Adducts and Oxidatively Induced DNA Damage in Aflatoxin-Treated Mice in Vivo as Measured by Liquid Chromatography-Tandem Mass Spectrometry with Isotope Dilution.

Dietary exposure to aflatoxin B1 (AFB1) is a significant contributor to the incidence of hepatocellular carcinomas globally. AFB1 exposure leads to the formation of AFB1-N7-guanine (AFB1-N7-Gua) and two diastereomers of the imidazole ring-opened 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxyaflatoxin B1 (AFB1-FapyGua) in DNA. These adducts lead to G → T transversion mutations with the ring-opened adduct being more mutagenic than the cationic species. Accurate measurement of these three adducts as biomarkers in DNA and urine will help identify dietary exposure to AFB1 as a risk factor in the development of hepatocellular carcinoma worldwide. Herein, we report an improved methodology for the measurement of AFB1-N7-Gua and the two diastereomers of AFB1-FapyGua using liquid chromatography-tandem mass spectrometry with isotope dilution. We measured the levels of these compounds in liver DNA of six control mice and six AFB1-treated mice. Levels varying from 1.5 to 45 lesions/106 DNA bases in AFB1-treated mice were detected depending on the compound and animal. No background levels of these adducts were detected in control mice. We also tested whether the AFB1 treatment caused oxidatively induced DNA base damage using gas chromatography-tandem mass spectrometry with isotope dilution. Although background levels of several pyrimidine- and purine-derived lesions were detected, no increases in these levels were found upon AFB1 treatment of mice. On the other hand, significantly increased levels of (5' R)- and (5' S)-8,5'-cyclo-2'-deoxyadenosines were observed in liver DNA of AFB1-treated mice. The impact of this work is expected to achieve the accurate measurement of three AFB1-DNA adducts and oxidatively induced DNA lesions as biomarkers of AFB1 exposure as germane to investigations designed for the prevention of aflatoxin-related hepatocellular carcinomas and for determining the effects of genetic deficiencies in human populations.

Excision release of 5?hydroxycytosine oxidatively induced DNA base lesions from the lung genome by cat dander extract challenge stimulates allergic airway inflammation.

BACKGROUND: Ragweed pollen extract (RWPE) induces TLR4-NFκB-CXCL-dependent recruitment of ROS-generating neutrophils to the airway and OGG1 DNA glycosylase-dependent excision of oxidatively induced 8-OH-Gua DNA base lesions from the airway epithelial cell genome. Administration of free 8-OH-Gua base stimulates RWPE-induced allergic lung inflammation. These studies suggest that stimulation of innate receptors and their adaptor by allergenic extracts initiates excision of a set of DNA base lesions that facilitate innate/allergic lung inflammation. OBJECTIVE: To test the hypothesis that stimulation of a conserved innate receptor/adaptor pathway by allergenic extracts induces excision of a set of pro-inflammatory oxidatively induced DNA base lesions from the lung genome that stimulate allergic airway inflammation. METHODS: Wild-type (WT), Tlr4KO, Tlr2KO, Myd88KO, and TrifKO mice were intranasally challenged once or repeatedly with cat dander extract (CDE), and innate or allergic inflammation and gene expression were quantified. We utilized GC-MS/MS to quantify a set of oxidatively induced DNA base lesions after challenge of naïve mice with CDE. RESULTS: A single CDE challenge stimulated innate neutrophil recruitment that was partially dependent on TLR4 and TLR2, and completely on Myd88, but not TRIF. A single CDE challenge stimulated MyD88-dependent excision of DNA base lesions 5-OH-Cyt, FapyAde, and FapyGua from the lung genome. A single challenge of naïve WT mice with 5-OH-Cyt stimulated neutrophilic lung inflammation. Multiple CDE instillations stimulated MyD88-dependent allergic airway inflammation. Multiple administrations of 5-OH-Cyt with CDE stimulated allergic sensitization and allergic airway inflammation. CONCLUSIONS AND CLINICAL RELEVANCE: We show for the first time that CDE challenge stimulates MyD88-dependent excision of DNA base lesions. Our data suggest that the resultant-free base(s) contribute to CDE-induced innate/allergic lung inflammation. We suggest that blocking the MyD88 pathway in the airways with specific inhibitors may be a novel targeted strategy of inhibiting amplification of innate and adaptive immune inflammation in allergic diseases by oxidatively induced DNA base lesions.

Biomarkers of oxidatively induced DNA damage in dreissenid mussels: A genotoxicity assessment tool for the Laurentian Great Lakes.

Activities of fast growing human population are altering freshwater ecosystems, endangering their inhabitants and public health. Organic and trace compounds have a high potential for adverse impacts on aquatic organisms in some Great Lakes tributaries. Toxic compounds in tissues of organisms living in contaminated environments change their metabolism and alter cellular components. We measured oxidatively induced DNA damage in the soft tissues of dreissenid mussels to check on the possible contaminant-induced impact on their DNA. The animals were obtained from archived samples of the National Oceanic and Atmospheric Administration (NOAA) Mussel Watch Program. Mussels were collected from the harbor of Ashtabula River in Ohio, and a reference area located at the Lake Erie shore. Using gas chromatography-tandem mass spectrometry with isotope dilution, we identified and quantified numerous oxidatively modified DNA bases and 8,5'-cyclopurine-2'-deoxynucleosides. We found significant differences in the concentrations of these potentially mutagenic and/or lethal lesions in the DNA of mussels from the harbor as compared to the animals collected at the reference site. These results align NOAA's data showing that elevated concentrations of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and heavy metals were found in mussels within the harbor as compared to mussels collected in the reference site. The measured DNA lesions can be used as biomarkers for identifying DNA damage in mussels from polluted and reference sites. Such biomarkers are needed to identify the bioeffects of contaminants in affected organisms, as well as whether remedial actions have proven successful in reducing observed toxic effects.

Exposure to Engineered Nanomaterials: Impact on DNA Repair Pathways.

Some engineered nanomaterials (ENMs) may have the potential to cause damage to the genetic material in living systems. The mechanistic machinery functioning at the cellular/molecular level, in the form of DNA repair processes, has evolved to help circumvent DNA damage caused by exposure to a variety of foreign substances. Recent studies have contributed to our understanding of the various DNA damage repair pathways involved in the processing of DNA damage. However, the vast array of ENMs may present a relatively new challenge to the integrity of the human genome; therefore, the potential hazard posed by some ENMs necessitates the evaluation and understanding of ENM-induced DNA damage repair pathways. This review focuses on recent studies highlighting the differential regulation of DNA repair pathways, in response to a variety of ENMs, and discusses the various factors that dictate aberrant repair processes, including intracellular signalling, spatial interactions and ENM-specific responses.

Extreme Expression of DNA Repair Protein Apurinic/Apyrimidinic Endonuclease 1 (APE1) in Human Breast Cancer As Measured by Liquid Chromatography and Isotope Dilution Tandem Mass Spectrometry.

Apurinic/apyrimidinic endonuclease 1 (APE1) is a DNA repair protein and plays other important roles. Increased levels of APE1 in cancer have been reported. However, available methods for measuring APE1 levels are indirect and not quantitative. We previously developed an approach using liquid chromatography and tandem mass spectrometry with isotope dilution to accurately measure APE1 levels. Here, we applied this methodology to measure APE1 levels in normal and cancerous human breast tissues. Extreme expression of APE1 in malignant tumors was observed, suggesting that breast cancer cells may require APE1 for survival. Accurate measurement of APE1 may be essential for the development of novel treatment strategies and APE1 inhibitors as anticancer drugs.

The comet assay as a tool for human biomonitoring studies: the ComNet project.

The comet assay is widely used in human biomonitoring to measure DNA damage as a marker of exposure to genotoxic agents or to investigate genoprotective effects. Studies often involve small numbers of subjects, and design may be sub-optimal in other respects. In addition, comet assay protocols in use in different laboratories vary significantly. In spite of these difficulties, it is appropriate to carry out a pooled analysis of all available comet assay biomonitoring data, in order to establish baseline parameters of DNA damage, and to investigate associations between comet assay measurements and factors such as sex, age, smoking status, nutrition, lifestyle, etc. With this as its major objective, the ComNet project has recruited almost 100 research groups willing to share datasets. Here we provide a background to this project, discussing the history of the comet assay and practical issues that can critically affect its performance. We survey its diverse applications in biomonitoring studies, including environmental and occupational exposure to genotoxic agents, genoprotection by dietary and other factors, DNA damage associated with various diseases, and intrinsic factors that affect DNA damage levels in humans. We examine in depth the quality of data from a random selection of studies, from an epidemiological and statistical point of view.

Could 8-oxoguanine DNA glycosylase 1 Ser326Cys polymorphism be a biomarker of susceptibility in cancer?

Biomarkers are key molecular or cellular events that give an indication whether there is a threat for disease, whether a disease already exists, or how such disease may develop in an individual case. The discovery of polymorphisms in genes that function in the metabolism of chemicals and in DNA repair has demonstrated the importance of understanding the phenomenon of genetic susceptibility in a population. Polymorphisms in DNA repair genes as an important component of the individual susceptibility to the development of cancer and various hereditary diseases have been commonly studied, since these genes have critical roles in maintaining genome integrity. Furthermore, the evaluation of cancer risk depends on the level of exposure to carcinogenic factors as well as on the genetic codes of the individual. This approach is supported by studies that present positive association between these polymorphic genes and cancers. Although 8-oxoguanine DNA glycosylase 1 (OGG1) is one of the promising biomarker candidates of cancer susceptibility, there are also some controversial results. Epidemiological studies show that the OGG1 might be a biomarker of susceptibility for various cancers; however, the small sample size and difference in the eligibility criteria for inclusion of subjects and sources might limit the studies to demonstrate the association between the OGG1 Ser326Cys polymorphism and the risk of cancer. Thus, meta-analyses may provide more valuable and reliable data to demonstrate the potential of OGG1 Ser326Cys DNA repair enzyme polymorphisms that could be the biomarkers of susceptibility of cancer. Our aim in this review is to compile published studies, including some controversial results on the association between the OGG1 Ser326Cys polymorphism and the risk of cancer.

Can linoleic acid improve the quality of frozen thawed bull sperm?

BACKGROUND: Cryopreservation is known to have a detrimental effect on the motility, viability and membrane integrity of sperm cells. OBJECTIVE: The aim of this study was to investigate the effect of various amount of linoleic acid supplementation to the Tris extender, on bull sperm parameters, DNA integrity and oxidative stress after freeze-thawing. METHODS: Ejaculates were split into five aliquots and extended to a final concentration of 18x10(6) spermatozoa per ml with the base extender containing different doses of linoleic acid 0.125 ml, (L125); 0.250 ml (L250); 0.5 ml (L500), 1 ml (L1000) and no additive (control; L0). The extended samples were equilibrated slowly to 4 degree C for 4 h and then froze using a digital freezing machine. Frozen straws were thawed individually in water bath at 37 degree C for 30 s to analyse progressive motility and sperm motion characteristics as well as membrane integrity. Biochemical assays were performed in a spectrophotometer using commercial kits. DNA damage was evaluated by Comet Assay. RESULT: The addition of various linoleic acid did not improve the sperm subjective, CASA and progressive motilities, sperm motility characteristics and DNA integrity (P>0.05). L500 exhibited the greatest values for membrane integrity than that of the other groups (P<0.001). All supplementation groups led to lower percentages of tail abnormalities in comparison to the control (P<0.001). L500 and L1000 significantly decreased total abnormalities. In conclusion, our findings showed that L500 linoleic acid supplementation in semen extender was of great beneficial effect on frozen-thawed bull semen in terms of morphology and plasma membrane integrity.

Effects of various cryoprotectants on bull sperm quality, DNA integrity and oxidative stress parameters.

The objectives of this study was to compare the effects of type and concentration of cryoprotectants glycerol (G), ethylene glycol (EG) and dimethyl sulfoxide (DMSO) on the plasma membrane and DNA integrity as well as antioxidant activity of cryopreserved Eastern Anatolian red bull sperm. Ejaculates were collected from the three bulls using an artificial vagina twice a week. The ejaculates were pooled to increase the semen volume for replication and to eliminate variability among the evaluated samples. The pooled ejaculates were also split into seven equal experimental groups and diluted with the modified base extender to a final spermatozoa concentration of 15×10(6)/ml. The extended samples were cooled slowly to 4°C and equilibrated for 4h. They were then loaded into 0.25ml French straws and frozen using a digital freezing machine at 3 programmed rates: -3°C/min from +4°C to -10°C, -40°C/min from -10°C to -100°C, and -20°C/min from -100°C to -140°C. Thereafter, the straws were plunged into liquid nitrogen at -196°C. Frozen straws were thawed individually at 37°C for 30s in a water bath to analyse progressive motility and sperm motion characteristics as well as membrane integrity using hypo-osmotic swelling test. Biochemical assays were performed in a spectrophotometer using commercial kits. DNA damage was evaluated by Comet Assay using Image Analysis System. 6% G exhibited the greatest percentages of CASA (43.7±2.92%) and progressive (26.4±2.64%) motilities when compared to the other groups (P<0.001). 6% G and 6% EG showed the greatest values of preserved membrane integrity (P<0.001). 6% DMSO and 3% EG + 3% DMSO resulted in greater chromatin damage than the other groups (P<0.001). The antioxidant activities of GPx, GSH, and CAT as well as the total antioxidant activity were affected by the type of cryoprotectant; notably, 2% G+2% EG+2% DMSO yielded the lowest activities when compared to the other groups (P<0.001). In conclusion, no advantages were found in using EG or DMSO to replace G in bull sperm cryopreservation. Freezing with cryoprotectant 6% G yielded the best post-thaw sperm characteristics for Eastern Anatolian Red bull spermatozoa.

Possible Genetic Risks from Heat-Damaged DNA in Food.

The consumption of foods prepared at high temperatures has been associated with numerous health risks. To date, the chief identified source of risk has been small molecules produced in trace levels by cooking and reacting with healthy DNA upon consumption. Here, we considered whether the DNA in food itself also presents a hazard. We hypothesize that high-temperature cooking may cause significant damage to the DNA in food, and this damage might find its way into cellular DNA by metabolic salvage. We tested cooked and raw foods and found high levels of hydrolytic and oxidative damage to all four DNA bases upon cooking. Exposing cultured cells to damaged 2'-deoxynucleosides (particularly pyrimidines) resulted in elevated DNA damage and repair responses in the cells. Feeding a deaminated 2'-deoxynucleoside (2'-deoxyuridine), and DNA containing it, to mice resulted in substantial uptake into intestinal genomic DNA and promoted double-strand chromosomal breaks there. The results suggest the possibility of a previously unrecognized pathway whereby high-temperature cooking may contribute to genetic risks.

Polymorphic variant Asp239Tyr of human DNA glycosylase NTHL1 is inactive for removal of a variety of oxidatively-induced DNA base lesions from genomic DNA.

Base excision repair is the major pathway for the repair of oxidatively-induced DNA damage, with DNA glycosylases removing modified bases in the first step. Human NTHL1 is specific for excision of several pyrimidine- and purine-derived lesions from DNA, with loss of function NTHL1 showing a predisposition to carcinogenesis. A rare single nucleotide polymorphism of the Nthl1 gene leading to the substitution of Asp239 with Tyr within the active site, occurs within global populations. In this work, we overexpressed and purified the variant NTHL1-Asp239Tyr (NTHL1-D239Y) and determined the substrate specificity of this variant relative to wild-type NTHL1 using gas chromatography-tandem mass spectrometry with isotope-dilution, and oxidatively-damaged genomic DNA containing multiple pyrimidine- and purine-derived lesions. Wild-type NTHL1 excised seven DNA base lesions with different efficiencies, whereas NTHL1-D239Y exhibited no glycosylase activity for any of these lesions. We also measured the activities of human glycosylases OGG1 and NEIL1, and E. coli glycosylases Nth and Fpg under identical experimental conditions. Different substrate specificities among these DNA glycosylases were observed. When mixed with NTHL1-D239Y, the activity of NTHL1 was not reduced, indicating no substrate binding competition. These results and the inactivity of the variant D239Y toward the major oxidatively-induced DNA lesions points to the importance of the understanding of this variant's role in carcinogenesis and the potential of individual susceptibility to cancer in individuals carrying this variant.

Inhibition of human APE1 and MTH1 DNA repair proteins by dextran-coated γ-Fe2O3 ultrasmall superparamagnetic iron oxide nanoparticles.

Aim: To quantitatively evaluate the inhibition of human DNA repair proteins APE1 and MTH1 by dextran-coated γ-Fe2O3 ultrasmall superparamagnetic iron oxide nanoparticles (dUSPIONs). Materials & methods: Liquid chromatography-tandem mass spectrometry with isotope-dilution was used to measure the expression levels of APE1 and MTH1 in MCL-5 cells exposed to increasing doses of dUSPIONs. The expression levels of APE1 and MTH1 were measured in cytoplasmic and nuclear fractions of cell extracts. Results: APE1 and MTH1 expression was significantly inhibited in both cell fractions at the highest dUSPION dose. The expression of MTH1 was linearly inhibited across the full dUSPION dose range in both fractions. Conclusion: These findings warrant further studies to characterize the capacity of dUSPIONs to inhibit other DNA repair proteins in vitro and in vivo.

Inhibitors of DNA repair proteins are increasingly being utilized as potential anticancer agents to supplement traditional chemotherapy and radiation-based approaches. The present study was focused on investigating the use of iron oxide nanoparticles to inhibit the expression of relevant human DNA repair proteins in a cellular model (MCL-5 cells). The authors utilized liquid chromatography­tandem mass spectrometry with isotope dilution to measure the expression levels of two different DNA repair proteins (MTH1 and APE1) in cells after the cells were exposed to increasing levels of the iron oxide nanoparticles. The authors observed significant decreases in DNA repair protein levels that were associated with increasing doses of the iron oxide nanoparticles. The authors' findings warrant more comprehensive studies using other cellular models and suitable animal models.

A pooled analysis of molecular epidemiological studies on modulation of DNA repair by host factors.

Levels of DNA damage represent the dynamics between damage formation and removal. Therefore, to better interpret human biomonitoring studies with DNA damage endpoints, an individual's ability to recognize and properly remove DNA damage should be characterized. Relatively few studies have included DNA repair as a biomarker and therefore, assembling and analyzing a pooled database of studies with data on base excision repair (BER) was one of the goals of hCOMET (EU-COST CA15132). A group of approximately 1911 individuals, was gathered from 8 laboratories which run population studies with the comet-based in vitro DNA repair assay. BER incision activity data were normalized and subsequently correlated with various host factors. BER was found to be significantly higher in women. Although it is generally accepted that age is inversely related to DNA repair, no overall effect of age was found, but sex differences were most pronounced in the oldest quartile (>61 years). No effect of smoking or occupational exposures was found. A body mass index (BMI) above 25 kg/m2 was related to higher levels of BER. However, when BMI exceeded 35 kg/m2, repair incision activity was significantly lower. Finally, higher BER incision activity was related to lower levels of DNA damage detected by the comet assay in combination with formamidopyrimidine DNA glycosylase (Fpg), which is in line with the fact that oxidatively damaged DNA is repaired by BER. These data indicate that BER plays a role in modulating the steady-state level of DNA damage that is detected in molecular epidemiological studies and should therefore be considered as a parallel endpoint in future studies.

Micronucleus frequencies in lymphocytes and buccal epithelial cells from patients having head and neck cancer and their first-degree relatives.

Head and neck squamous cell carcinoma is the fifth most common cancer type worldwide. Even though it is known that the most important environmental aetiological factors for head and neck cancer (HNC) development are tobacco and alcohol, genetic susceptibility is also thought to be important. The use of biomarkers of chromosomal damage due to genetic instability in order to predict risk of cancer as well as to identify high-risk individuals is imperative. We have investigated genetic damage in patients having HNC (n = 59) and their first-degree relatives (FDRs) (n = 34) with a biomarker in two different tissues; the micronucleus (MN) test in peripheral blood lymphocytes and in exfoliated buccal cells. The mean (standard deviation) levels of MN frequencies (‰) in lymphocytes of patients, relatives and controls were 27.10 (9.52), 14.09 (5.13) and 9.00 (6.87), respectively. The mean (standard deviation) levels of MN frequencies (‰) in exfoliated buccal cells of patients, relatives and controls were 2.87 (1.16), 1.38 (0.85) and 1.23 (0.93), respectively. Our results indicated that spontaneous genetic damage in lymphocytes of patients having HNC was significantly higher than that of controls (P < 0.01) and thus genetic instability appeared to exist in lymphocytes of cancer patients. Similar findings were obtained for exfoliated buccal cell MN frequencies of cancer patients (P < 0.01). We observed that the FDRs of patients having HNC showed significantly higher chromosomal damage in terms of MN frequencies in lymphocytes when compared with those of controls (P < 0.05), thus reflecting an increased susceptibility to HNC in FDRs. However, for buccal cell MN frequencies, we could not demonstrate enhanced genetic instability in the FDRs of patients having HNC.

The HUman MicroNucleus project on eXfoLiated buccal cells (HUMN(XL)): the role of life-style, host factors, occupational exposures, health status, and assay protocol.

The human buccal micronucleus cytome assay (BMCyt) is one of the most widely used techniques to measure genetic damage in human population studies. Reducing protocol variability, assessing the role of confounders, and estimating a range of reference values are research priorities that will be addressed by the HUMN(XL) collaborative study. The HUMN(XL) project evaluates the impact of host factors, occupation, life-style, disease status, and protocol features on the occurrence of MN in exfoliated buccal cells. In addition, the study will provide a range of reference values for all cytome endpoints. A database of 5424 subjects with buccal MN values obtained from 30 laboratories worldwide was compiled and analyzed to investigate the influence of several conditions affecting MN frequency. Random effects models were mostly used to investigate MN predictors. The estimated spontaneous MN frequency was 0.74‰ (95% CI 0.52-1.05). Only staining among technical features influenced MN frequency, with an abnormal increase for non-DNA-specific stains. No effect of gender was evident, while the trend for age was highly significant (p<0.001). Most occupational exposures and a diagnosis of cancer significantly increased MN and other endpoints frequencies. MN frequency increased in heavy smoking (≥40cig/day, FR=1.37; 95% CI 1.03-.82) and decreased with daily fruit consumption (FR=0.68; 95% CI 0.50-0.91). The results of the HUMN(XL) project identified priorities for validation studies, increased the basic knowledge of the assay, and contributed to the creation of a laboratory network which in perspective may allow the evaluation of disease risk associated with MN frequency.

Inhibition by Tetrahydroquinoline Sulfonamide Derivatives of the Activity of Human 8-Oxoguanine DNA Glycosylase (OGG1) for Several Products of Oxidatively induced DNA Base Lesions.

DNA glycosylases involved in the first step of the DNA base excision repair pathway are promising targets in cancer therapy. There is evidence that reduction of their activities may enhance cell killing in malignant tumors. Recently, two tetrahydroquinoline compounds named SU0268 and SU0383 were reported to inhibit OGG1 for the excision of 8-hydroxyguanine. This DNA repair protein is one of the major cellular enzymes responsible for excision of a number of oxidatively induced lesions from DNA. In this work, we used gas chromatography-tandem mass spectrometry with isotope-dilution to measure the excision of not only 8-hydroxyguanine but also that of the other major substrate of OGG1, i.e., 2,6-diamino-4-hydroxy-5-formamidopyrimidine, using genomic DNA with multiple purine- and pyrimidine-derived lesions. The excision of a minor substrate 4,6-diamino-5-formamidopyrimidine was also measured. Both SU0268 and SU0383 efficiently inhibited OGG1 activity for these three lesions, with the former being more potent than the latter. Dependence of inhibition on concentrations of SU0268 and SU0383 from 0.05 µmol/L to 10 µmol/L was also demonstrated. The approach used in this work may be applied to the investigation of OGG1 inhibition by SU0268 and SU0383 and other small molecule inhibitors in further studies including cellular and animal models of disease.

Ne-22 Ion-Beam Radiation Damage to DNA: From Initial Free Radical Formation to Resulting DNA-Base Damage.

We report on the physicochemical processes and the products of DNA damage involved in Ne-22 ion-beam radiation of hydrated (12 ± 3 H2O/nucleotide) salmon testes DNA at 77 K. Free radicals trapped at 77 K were identified using electron spin resonance (ESR) spectroscopy. The measurement of DNA damage using two different techniques of mass spectrometry revealed the formation of numerous DNA products. Results obtained by ESR spectroscopy showed that as the linear energy transfer (LET) of the ion-beam radiation increases along the beam track, the production of DNA radicals correspondingly increases until just before the Bragg peak is reached. Yields of DNA products along the ion-beam track were in excellent agreement with the radical production. This work is the first to use the combination of ESR spectroscopy and mass spectrometric techniques enabling a better understanding of mechanisms of radiation damage to DNA by heavy ion beams detailing the formation of DNA free radicals and their subsequent products.

DNA glycosylase deficiency leads to decreased severity of lupus in the Polb-Y265C mouse model.

The Polb gene encodes DNA polymerase beta (Pol ß), a DNA polymerase that functions in base excision repair (BER) and microhomology-mediated end-joining. The Pol ß-Y265C protein exhibits low catalytic activity and fidelity, and is also deficient in microhomology-mediated end-joining. We have previously shown that the PolbY265C/+ and PolbY265C/C mice develop lupus. These mice exhibit high levels of antinuclear antibodies and severe glomerulonephritis. We also demonstrated that the low catalytic activity of the Pol ß-Y265C protein resulted in accumulation of BER intermediates that lead to cell death. Debris released from dying cells in our mice could drive development of lupus. We hypothesized that deletion of the Neil1 and Ogg1 DNA glycosylases that act upstream of Pol ß during BER would result in accumulation of fewer BER intermediates, resulting in less severe lupus. We found that high levels of antinuclear antibodies are present in the sera of PolbY265C/+ mice deleted of Ogg1 and Neil1 DNA glycosylases. However, these mice develop significantly less severe renal disease, most likely due to high levels of IgM in their sera.

Increased micronucleus frequencies in surrogate and target cells from workers exposed to crystalline silica-containing dust.

Mining, crushing, grinding, sandblasting and construction are high-risk activities with regard to crystalline silica exposure, especially in developing countries. Respirable crystalline silica (quartz and cristobalite) inhaled from occupational sources has been reclassified as a human carcinogen in 1997 by the International Agency for Research on Cancer. However, the biological activity of crystalline silica has been found to be variable among different industries, and this has formed the basis for further in vivo/in vitro mechanistic research and epidemiologic studies. This study was conducted for genotoxicity evaluation in a population of workers (e.g. glass industry workers, sandblasters, and stone grinders) mainly exposed to crystalline silica in four different workplaces in Turkey. The micronucleus (MN) assay was applied both in peripheral blood lymphocytes (PBL) as a surrogate tissue and in nasal epithelial cells (NEC) as a target tissue of the respiratory tract. Our study revealed significantly higher MN frequencies in the workers (n = 50) versus the control group (n = 29) (P < 0.001) and indicated a significant effect of occupational exposure on MN induction in both of the tissues. For the NEC target tissue, the difference in MN frequencies between the workers and control group was 3-fold, whereas in peripheral tissue, it was 2-fold. Respirable dust and crystalline silica levels exceeding limit values and mineralogical/elemental dust composition of the dust of at least 70% SiO(2) were used as markers of crystalline silica exposure in each of the workplaces. Moreover, 24% of the current workers were found to have early radiographical changes (profusion category of 1). In conclusion, although the PBL are not primary target cells for respiratory particulate toxicants, an evident increase in MN frequencies in this surrogate tissue was observed, alongside with a significant increase in NEC and may be an indicator of the accumulated genetic damage associated with crystalline silica exposure.